1. Field of the Invention
This invention relates to hydroxyapatite resins and their use in protein elutions.
2. Description of the Prior Art
Hydroxyapatite is known to be useful in the separation and purification of proteins using retention protocols that involve affinity, ion exchange or hydrophobic interactions. Hydroxyapatite is particularly useful in the purification of recombinant proteins from host cell proteins, aggregates, endotoxin, and DNA. Protein loading of a hydroxyapatite column is commonly conducted at pH 6.5 with phosphate buffer at 2 mM to 5 mM, conditions that promote the adsorption of protein to the hydroxyapatite surface. In some cases, adsorption is further promoted by the inclusion of minor amounts of NaCl or KCl. Prior to protein loading, the resin is commonly equilibrated with a buffer of the same strength as the loading buffer and at the same pH. The equilibration and loading buffers both saturate the hydroxyapatite surface with hydroxonium ions (H3O+). Unfortunately, these ions tend to desorb during protein elution due to the acidic conditions that are typically encountered during the elution, and this causes the resin to deteriorate over time.